Arrays of electrodes coated with molecules and their production

ABSTRACT

The present invention provides a method of forming coatings of at least two different coating molecules on at least two electrodes, the method comprising: (a) providing an array of at least two individually-addressable electrodes, (b) allowing a layer of a masking molecule to adsorb onto all electrodes, (c) inducing electrochemical desorption of the masking molecule from at least one but not all electrodes to expose a first set of exposed electrodes, (d) allowing a first coating molecule to adsorb onto the first set of exposed electrodes, (e) exposing all electrodes to a masking molecule to allow adsorption of the masking molecule onto all electrodes, (f) inducing electrochemical desorption of masking molecule from a second set of electrodes to expose a second set of exposed electrodes, (g) allowing a second coating molecule to adsorb onto the second set of exposed electrodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/GB2003/004368, filed Oct. 9, 2003, which claims the benefit ofGreat Britain Patent Application No. 0223666.9, filed Oct. 10, 2002,which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to methods for production of arrays of a number ofdifferent coating molecules by forming coatings on a number of differentelectrodes. It also relates to arrays of molecules produced by thesemethods.

BACKGROUND OF THE INVENTION

Among the many challenges facing the development of a molecular-basednanotechnology, the directed assembly of discrete molecular objects, andtheir controlled integration into macroscopic structures, arefundamental. The selective self-assembly characteristic inherent tocertain molecules (for example, the Watson-Crick specific base pairingthat occurs between complementary single strands of DNA) is a propertythat could be exploited to address these challenges. For example,ordered suspensions of gold nanoparticles have been assembled by firstfunctionalizing the nanoparticles with short DNA oligonucleotides andthen introducing complementary DNA to tie the individual particlestogether as the strands hybridise (Mirkin et al, “A DNA-based method forrationally assembling nanoparticles into macroscopic materials”, Nature382, 607-609 (1996)). In principle, this concept could be employed totackle the integration of nanoscale elements onto a macroscopicsubstrate, such as an array of metal electrodes. Providing eachelectrode is functionalized with anchoring oligonucleotides of a uniquesequence, the nanoscale elements will assemble appropriately if they arefunctionalized with the complementary oligonucleotides.

Indeed, the ability to pattern a surface locally with differentmolecular monolayers in a well-controlled fashion and with a highspatial resolution has importance for molecular electronics andbiotechnology applications (including high density DNA expressionanalysis and genotyping), as well as for nanoengineering.

However, the selective multiple coating of electrode arrays withappropriate anchor molecules has not yet been demonstrated on structureswhere the electrode separations are sub-micrometre, limiting theapplicability of this procedure for nanoscale assembly.

A number of techniques are available for introducing oligonucleotides orother anchor molecules locally onto a surface, but none of thesesimultaneously meet the requirements of resolution, speed and theability to coat different electrodes uniquely. Microdrop dispensingsystems provide simple approaches for the controlled multiple coating ofan electrode array, but are restricted to a spatial resolution ofgreater than 10 □m. Micromachining and microcontact printing offerspatial resolutions of several hundred nanometres but lack the abilityto effect multiple coating. A much higher resolution (a few tens ofnanometres) has been achieved by nanografting, but this technique isslow, lacks a straightforward extension to allow multiple coating, andrequires complex and expensive infrastructure. Recently, a variant ofthe nanografting technique, dip-pen nanolithography, has been reported.This technique uses an AFM (atomic force microscope) tip, coated withthe anchor molecules, as a pen to draw onto the surface. The resolutionof this coating technique is also on the nanometre scale, but a highlevel of stability and solubility of the anchor molecules is required.

It is known that monolayers of thiol compounds can be formed on a goldsurface by immersing the surface in an aqueous solution containing thethiol molecule of interest. The gold-sulphur bond formed during thisspontaneous chemisorption process can undergo reductive cleavage atabout −1 V versus a Ag/AgCl reference electrode, leading toelectrochemical desorption. Compounds having different functionalitiescan also form monolayers on gold surfaces and undergo electrochemicaldesorption. Similarly, monolayers of molecules having other functionalgroups can assemble on other surfaces, from which electrochemicaldesorption is also possible.

Electrochemical desorption has been applied by Wilhelm et al in“Patterns of functional proteins formed by local electrochemicaldesorption of self-assembled monolayers”, Electrochimica Acta., vol 47,No. 1, 2001/September, pages 275-281. The authors form an alkanethiolate monolayer on a gold electrode and use a scanningelectrochemical microscope (SECM) to induce local electrochemicaldesorption at defined regions of the alkane thiolate monolayer by usingan ultramicroelectrode (UME) of 10 μm diameter placed about 5 μm abovethe macroscopic SAM-covered gold electrode. The exposed regions are thenable to chemisorb an ω-functionalised thiol or disulphide such ascystamine. Functional proteins can then be coupled to the amino groupspresent in the modified regions of the monolayer. Because this methodrelies on desorption from regions of one large electrode using an UMEabove the electrode it is subject to resolution restrictions and indeedis restricted to a spatial resolution of around 10 μm. It means that itcan be difficult to control the system so that desorption is not inducedat areas neighbouring the intended area for desorption.

A different technique is described by Tender et al in “Electrochemicalpatterning of self-assembled monolayers onto microscopic arrays of goldelectrodes fabricated by laser ablation”, Langmuir, 1996, 12, 5515-5518.This group describe use of an array of individually-addressable goldmicroelectrodes. One technique involves adsorption of a monolayer of(1-mercaptoundec-11-yl) hexa(ethylene glycol) (EG₆SH) on all electrodes.Electrochemical desorption is then induced from alternating bands ofelectrodes, by controlling the potential at the electrodes from whichdesorption is required. Adsorption of a layer of hexadecanethiol (C₁₆SH)is then allowed to adsorb onto the thus-exposed bands. Thus, alternatingbands of C₁₆S and EG₆S monolayers are obtained. Non specific absorptionof BSA-antibody onto the C₁₆S bands is then allowed and BSA bindsspecifically to the antibody.

It is stated that the extension of electrochemical desorption of SAMs topattern SAMs of n different ω-substituted alkane thiols onto nindividually-addressable microscopic gold elements “should also bestraightforward”. However, we are not aware of any further publicationsby this group along these lines. Furthermore, we believe that thesuggested sequential stripping of the EG₆S SAM from different elementsand exposure to new alkane thiols, using the method described by Tenderet al, would result in contamination of previously patterned layers withsubsequently introduced alkane thiols.

Therefore it would be desirable to provide a method for the formation ofan array of two or more different molecules, the method being capable ofgiving nanoscale resolution (distance between areas coated withdifferent molecules) and high purity of the individually patternedregions. It would also be desirable to provide such a method which canbe carried out conveniently and at high speed.

SUMMARY OF THE INVENTION

According to the invention we provide a method of forming coatings of atleast two different coating molecules on at least two electrodes, themethod comprising:

-   -   (a) providing an array of at least two individually-addressable        electrodes,    -   (b) allowing a layer of a masking molecule to adsorb on to all        the electrodes,    -   (c) inducing electrochemical desorption of the masking molecule        from at least one but not all electrodes to expose a first set        of exposed electrodes,    -   (d) allowing a first coating molecule to adsorb onto the first        set of exposed electrodes,    -   (e) exposing all electrodes to a masking molecule to allow        adsorption of the masking molecule onto all electrodes,    -   (f) inducing electrochemical desorption of masking molecule from        a second set of electrodes to expose a second set of exposed        electrodes,    -   (g) allowing a second coating molecule to adsorb onto the second        set of exposed electrodes.

We find that the process has the advantage of allowing formation ofarrays of a large number of different coating molecules coated with highpurity at nanoscale resolution. In particular, we find that use of anarray of individually-addressable electrodes has significant advantagesover the method described by Wilhelm et al (which induces desorptionfrom different regions of a single electrode) that resolution can begreater and effects on regions neighbouring the electrodes can becontrolled to avoid unwanted desorption.

Step (e) is particularly important in the invention. This is areprotection step in which adsorption of a masking molecule is allowedto take place onto all electrodes, including the electrodes providedwith a layer of masking molecule and those onto which adsorption of thecoating molecule has occurred. We find that this step, not used orsuggested by Tender et al, prevents adsorption of subsequent coatingmolecules onto electrodes already coated by coating molecules in latersteps and minimises contamination. This allows the provision of largenumbers of highly pure coatings of different coating molecules.

We find that by the invention we can for the first time produce an arrayof electrodes having nanoscale separation coated with different coatingmolecules. Therefore in a second aspect we provide an array of at least3, preferably at least 5, more preferably at least 10 sets ofindividually-addressable electrodes, each set having adsorbed thereon adifferent coating molecule, the minimum distance between electrodesbeing not more than 900 nanometres, preferably not more than 100nanometres, more preferably not more than 50 nanometres.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows CV traces of clean, coated and exposed electrodes.

FIG. 2 shows an electrode array from which a masking molecule has beenselectively desorbed.

FIG. 3 is an SEM picture of the region between two electrodes accordingto the method of the invention.

FIG. 4 shows two further electrode arrays coated by the method of theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method of the invention requires the use of an array of at least twoindividually-addressable electrodes. Such arrays are known and can bemade in known manner. One method is described below. Preferably thearray comprises at least 10, more preferably at least 20, particularlypreferably at least 50 individually-addresssable electrodes.

Although the method of the invention is applicable to electrodes on anyscale, it is particularly beneficial for providing arrays of smallelectrodes, particularly of diameter (largest dimension) in the micronrange (e.g. below 50 μm), preferably on a nanoscale, namely at nanometreresolution. Thus preferably the electrodes have a diameter of not morethan 900 nanometres, preferably not more than 500 nanometres, morepreferably not more than 100 nanometres.

A particular benefit of the invention is the fact that it allows coatingof closely spaced electrodes. The minimum distance between neighbouringelectrodes can be below 80 μm, preferably below 30 μm and even below 10μm. Preferably the minimum distance between neighbouring electrodes isnot more than 900 nanometres, more preferably not more than 500nanometres, even more preferably not more than 100 nanometres, mostpreferably not more than 50 nanometres. The method of the invention caneven be applied when the separation between electrodes is less than 25nanometres. This separation preferably applies to the minimum distancebetween all neighbouring electrodes.

The electrodes are formed of electrically conductive material.Preferably they are metallic but can for instance be non-metallic suchas carbon or silicon electrodes. Gold, silver, platinum, copper andaluminium, in particular gold, are preferred.

Generally all electrodes are formed from the same material, although itis possible in the invention that some electrodes are formed from onematerial and one or more additional sets of electrodes are formed fromdifferent materials.

In step (b) of the method a layer of masking molecule is adsorbed ontoall electrodes, preferably after cleaning the electrodes in standardmanner. One suitable cleaning method is described below.

The masking molecule is adsorbed onto the surface of the electrodes.Thus it must be capable of adsorbing onto a surface formed of thematerial from which the electrodes are formed. Preferably the moleculecontains a functional group capable of forming a bond with the electrodesurface so as to form a monolayer of the masking molecule. Of course,the electrodes must be chosen so that the material from which they areformed is such that molecules exist which can form bonds with it.

A variety of molecule types which can form bonds with a variety ofsurfaces are known. Examples of masking molecules are thiolatedmolecules which can form bonds with, and hence adsorb onto gold andsilver and other metal surfaces, and silicon surfaces. A particularlypreferred combination is gold electrodes and a thiolated maskingmolecule. Suitable thiolated masking molecules are known and includesubstituted and unsubstituted alkane thiols. Suitable substituentsinclude hydroxyl. Other masking molecules are those having alkyl groupswhich can absorb onto silicon electrodes.

The masking molecule chosen should be electrochemically active incombination with the material from which the electrode is formed. Itshould be capable of adsorbing and electrochemically desorbing underconvenient conditions, taking into account the nature of the coatingmolecule also to be used in the method. It should be chosen so that itundergoes electrochemical desorption at a convenient potential, inparticular at a potential which is not so high that it results in damageto the coating molecules also used in the method. For instance,preferred masking molecules undergo electrochemical desorption from thechosen electrode at a potential in the range −10V to +10V, preferably−5V to +5V, more preferably −2V to +2V. Masking molecules which undergoelectrochemical desorption at a negative potential are preferred.

Generally the masking molecule will have relatively low molecularweight, for instance below 500, in particular below 200 or 150. Smallermasking molecules are preferred as these are more effective atreprotection in the reprotection step (e) discussed further below. Theyalso form a dense monolayer, which is advantageous.

The masking molecule is generally provided in solution, preferablyaqueous solution. Concentrations may be chosen as convenient and canrange for instance from 0.1 millimolar to 10 millimolar, preferably from0.5 millimolar upwards.

The electrodes are contacted with the masking molecule for a timeappropriate to allow adsorption of the molecule onto the electrodesurfaces. The time required will depend upon the precise conditions butis generally not more than 180 mins, preferably not more than 90 mins.

After the adsorption step (b) all electrodes will be provided with amonolayer of the masking molecule.

Step (c) in the method requires inducing electrochemical desorption ofthe masking molecule from a predetermined set of electrodes. This iscarried out in known manner by controlling the potential at the relevantelectrodes.

A first set of electrodes is chosen for desorption of the first maskingmolecule. At least one but not all of the electrodes are treated in thismanner. They form the first set of exposed electrodes.

The electrodes from which electrochemical desorption is not required aregenerally held at open circuit. However, the potential at some or all ofthese other electrodes can be controlled to counteract any possibleeffects on them from electrodes from which desorption is required,especially when these are neighbouring electrodes. This is particularlyuseful when the minimum distance between electrodes is 20 nm or less.

Desorption can be carried out for any appropriate duration to allow alldesired molecules to desorb. Preferably desorption time is not more than300 secs, more preferably not more than 240 secs.

Desorption can be carried out under the influence of AC or DC voltage.If AC voltage is chosen then the amplitude and frequency are chosen soas to ensure that the adsorbed masking molecules are subject to theelectrochemical desorption potential for a sufficient period of time toallow electrochemical desorption to occur.

In step (d) a first coating molecule is allowed to adsorb onto the firstset of exposed electrodes. This can be a small molecule, for instance ofmolecular weight not more than 500. However, in this case it preferablyis a molecule capable of binding a macromolecule such as a polypeptideafter the coating step. For instance it can be an amino-terminatedmolecule capable of binding a protein.

Preferably however the coating molecule is a macromolecule. Thus itpreferably has molecular weight at least 800, preferably at least 1000,more preferably at least 1500, most preferably at least 3000.

Preferred types of macromolecule are oligonucleotides (e.g. 5 to 150bases). These can be used to form arrays of a large number of differentstrands of DNA (DNA chips) e.g. for use in gene screening or for use asanchoring nucleotides for inducing directed assembly of nanoscaleelements functionalised with complementary oligonucleotides, e.g. foruse in molecular electronics.

Alternative macromolecules are polypeptides, including proteins such asenzymes. These can be used, for instance, in biosensor applications.Proteins and oligopeptides can be used in nanoscale assemblyapplications.

The coating molecule is capable of adsorbing onto the material fromwhich the electrodes are formed. Thus they can be functionalised with afunctional group capable of adsorbing. Any suitable functional group canbe used provided it is compatible with the electrode material. Forinstance, thiolated coating molecules are preferred, in particular whenthe electrodes are gold or silver, particularly gold.

The first coating molecule is generally provided in solution, preferablyaqueous solution. Concentration can be chosen as appropriate but isgenerally in the range 1 to 100 micromolar, preferably 5 to 50micromolar.

Adsorption times are generally in the ranges given above for adsorptionof the first masking molecule.

In some cases it can be preferred to subject oligonucleotide and othercoating molecules to an electric field during the adsorption step (d) inorder to induce appropriate orientation. It has been shown that DNAmolecules experience a dielectrophoretic force and an orientationaltorque in a non-uniform electric field as a result of the interactionbetween the induced dipole in the DNA and the electric field. The torqueand the dielectrophoretic force are a function of the magnitude and thefrequency of the applied electric field. In the invention this effectcan be used to assist and accelerate the adsorption process. Themolecules are attracted to the electrodes and oriented into the rightspatial orientation for adsorption when a suitable electric field isapplied. This applied field can be an AC or a DC field. AC field ispreferred.

This process can also be utilised during the electrochemical desorptionsteps. The electric field applied in order to induce orientation can besuper imposed onto the voltage applied to induce electrochemicaldesorption.

In some embodiments it is preferred to allow passive adsorption but insome embodiments it can be preferred to apply an electrochemicalpotential (AC or DC) to the electrodes in order to accelerate theadsorption process (i.e. active adsorption). Adsorption rates can insome cases be up to two orders of magnitude higher than in passiveadsorption, that is adsorption without application of anyelectrochemical potential. Electrochemical assistance of this type, whenused, is preferably applied to step (d), namely the adsorption of thefirst (and subsequent) coating molecule onto the electrodes, but canalso be applied to the adsorption of the masking molecule.

The electrochemical desorption (c) and the adsorption step (d) arepreferably carried out sequentially, namely desorption is completedprior to contact of the exposed electrodes with the coating molecule.However, in some applications it can be preferred to induceelectrochemical desorption of the masking molecule when the electrodesare in contact with the coating molecules so that the two steps can takeplace simultaneously.

Step (e) in the method of the invention is the reprotection step. Allelectrodes are exposed to a second masking molecule so that the secondmasking molecule can be adsorbed onto all electrodes. This secondmasking molecule can be selected from any of those discussed above forthe masking molecule and preferably is the same molecule as is used instep (b). Conditions for this adsorption step can be selected from thosediscussed above in connection with step (b).

During this step it is believed that the masking molecule adsorbs ontothe just-coated electrodes between the coating molecules. Generally, themonolayer of coating molecules has not completely covered the electrodesand the monolayer of coating molecules is somewhat discontinuous. Themasking molecules thus protect these electrodes and prevent adsorptionof different coating molecules onto these electrodes in future steps.

Steps (f) and (g) essentially consist of repeating steps (c) and (d) fora second set of electrodes and a second coating molecule.

The second coating molecule is preferably selected from the same classof molecules as discussed above for the first coating molecule. It is adifferent molecule from the first coating molecule.

Steps (c) to (e) are then repeated as desired to form further sets ofexposed electrodes, and expose these sets to further masking molecule.

Thus the method of the invention can be used to provide any number ofsets of exposed electrodes carrying the same number of different coatingmolecules. In some cases an array can be produced in which everyelectrode is coated with a different coating molecule.

Examples

A series of opposing gold electrodes of sub-50 nm-separation wasfabricated on a Si/SiO₂ wafer using known UV lithography/lift offtechniques as described below. The wafer was cleaned by washing in‘piranha etch’ (30% H₂O₂, 70% H₂SO₄) for 1 hour, and then thoroughlyrinsed in deionised water, ethanol, and again in deionised water. Theentire electrode array was then coated with a protective molecularmonolayer of 6-mercapto-1-hexanol (MCH) by immersing the wafer in a 1 mMaqueous solution of MCH for 60 min. FIG. 1 compares the CV trace of acoated electrode (solid line) with that of a clean electrode prior tocoating (dashed line). A reductive desorption feature observed at around−1 V for the coated electrode indicates the removal of the MCHmonolayer. All electrochemical measurements were performed in 100 mMphosphate buffer at pH 10 using a standard three electrode setup at arate of 62 mV/s. A high purity platinum wire was used as the counterelectrode. All electrochemical potentials are reported vs. a Ag/AgClreference electrode.

To obtain complete desorption of the MCH monolayer from a particularelectrode, an electrochemical potential of −1.4 V vs. Ag/AgCl wasapplied to the electrode for two minutes while keeping all otherelectrodes at open circuit. FIG. 1 shows the CV trace after thisprocedure (dotted line), which, when compared with the trace for theclean surface, demonstrates that the monolayer on this particularelectrode was removed. The other electrodes, which were kept at opencircuit during the desorption step, were not affected and their CVtraces remained similar to the solid line in FIG. 1. (not shown). Thelarge increase in current observed in all traces below −1.2 V isassociated with hydrogen evolution.

In order to demonstrate that the MCH monolayer can act as a molecularmask, FIG. 2 shows an electrode array from which the MCH was selectivelydesorbed from electrodes numbered 2, 4 and 6. Thiofated oligonucleotidesX of sequence CAGGATGGCGAACAACAAGA-thiol (the thiol is connected to theoligonucleotide via a carbon C₆-linker) were dissolved in 10 mMtris(hydroxymethyl)aminomethane, 1 mM EDTA and 1 M NaCl solution of pH 8to a final concentration of 10 W. The array; with the MCH molecular masknow covering only electrodes 1, 3 and 5, was then immersed in thisaqueous solution for 60 min to allow the oligonucleotides to chemisorbto the exposed electrodes.

In order to detect the bound oligonucleotides, and to show that theyretain their selective self-assembly properties, a solution ofbiotinylated oligonucleotide X of sequence TCTTGTTGTTCGCCATCCTG-biotin(complementary to X) was applied to the electrode array for 90 min toallow the biotinylated oligonucleotides to hybridise to thesurface-bound oligonucleotide monolayers. Using an anti-biotin antibodydetection procedure described below, the presence of the biotin label(and hence the thiolated oligonucleotides) can be detected via a localcolour darkening. FIG. 2 shows that this occurs on electrodes 2, 4 and 6from which the MCH monolayer was removed. We note that the gap betweenopposing electrodes is too small to be resolved by optical microscopy inFIG. 2 but its presence can be inferred from the colouring of theelectrodes and the abrupt change in colour across the designed locationof the gap. A SEM picture of the region between electrodes 1 and 4 isshown in FIG. 3; the shortest distance between the electrodes isconsiderably less than 50 nm. The other electrode pairs were separatedby similar sized gaps (not shown).

FIG. 4 shows two electrode arrays on which electrodes 1, 3 and 5 werecoated with thiolated oligonucleotide Y (AGGTCGCCGCCC-thiol) and thenimmersed in 1 mM MCH for 60 min to strengthen the protectioncapabilities of the oligonucleotide monolayers. Next, the MCH remainingon electrodes 2, 4 and 6 of both arrays was desorbed to allow coatingwith thiolated oligonucleotide X. This coating step does notsignificantly affect existing MCH-oligonucleotide monolayers since theexchange rate between two thiolated oligonucleotides of similar length,one of which is bound to a gold surface, is expected to be very small.Subsequently, both arrays were again immersed in 1 mM MCH for 60 min,which does not significantly affect the oligonucleotide densities onelectrodes already coated with mixed oligonucleotide monolayers. Thearrays were then challenged with different biotinylated oligonucleotidesfor 90 mins: the array in FIG. 4( a) was challenged with biotinylatedoligonucleotide Y (of sequence GGGCGGCGACCT-biotin, complementary to Y),the array in FIG. 4( b) with biotinylated oligonucleotide X. The colourchange resulting from subsequent detection with the anti-biotin antibodyprocedure confirms that the thiolated oligonucleotides X and Y bound tothe desired electrodes and demonstrates that this technique can be usedto deposit different oligonucleotides selectively onto sub-50nm-separated electrodes. We note that the anti-biotin antibody detectionnot only shows that the required coating has been achieved but also thatthe bound thiolated oligonucleotides, which could act as anchormolecules in nanoassembly applications, remain intact and can stillhybridise with their complementary counterparts.

Fabrication of Electrode Array.

The electrode array was fabricated on a Si/SiO₂ wafer using a two-stepshadow evaporation technique (Philipp, G., Weimann, T., Hinze, P.,Burghard, M. & Weis, J., “Shadow evaporation method for fabrication ofsub 10 nm gaps between metal electrodes”, Microelectron. Eng. 46,157-160 (1999)). In the first step, a series of opposing electrodes ofseparation 35 μm comprising a 35-nm-thick Au layer on top of a 10 nmadhesive layer of Ni/Cr was created by standard UV photolithography,metal evaporation, and lift-off. In the second step, the wafer wastilted appropriately in the evaporator and 5 nm of Ni/Cr followed by 17nm of Au was deposited in stripes connecting the opposing electrodes.However, because the wafer was tilted, the edges of the existingelectrodes closest to the evaporation source shadowed the surface fromthe evaporation beam leading to the formation of sub-50-nm sized gapsbetween opposite electrodes.

DNA Detection

The protocol employed to visualise a specific oligonucleotide monolayerformed on a particular electrode of the arrays is based on acalorimetric detection of oligonucleotide hybridisation. Biotinylatedoligonucleotides of sequence complementary to the thiolatedoligonucleotides X and Y (X: TCTTGTTGTTCGCCATCCTG-biotin and Y:GGGCGGCGACCT-biotin) were dissolved in 10 mMtris(hydroxymethyl)aminomethane, 1 mM EDTA (TE solution) and 1 M NaCl toa final concentration of 2.5 μM. The appropriate biotinylatedoligonucleotide solution was then applied to the electrode array for 90min at room temperature to hybridise onto the complementarysurface-bound thiolated oligonucleotides. The biotinylatedoligonucleotide solution was rinsed off in tris-buffered saline (TBS)and, after several further washing steps, the electrode array wasimmersed in a 1:1000 dilution of monoclonal anti-biotin antibodyconjugated with alealine phosphatase in TBS/Tween 20 for 60 min.Immersing the electrode array in a solution of5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium causes alocal colour darkening where alkaline phosphatase is present, andtherefore where the biotinylated oligonucleotides are hybridised to theelectrode array. All oligonucleotides were purchased from MWG BiotechAG; all other reagents were purchased from Sigma.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1: Cyclic voltammograms of a bare Au electrode immediately aftercleaning (dashed line), the same electrode after coating with a MCHmolecular monolayer (solid line), and after desorbing the MCH monolayer(dotted line). All voltammograms were measured at 62 mV/s in 100 mMphosphate buffer at pH 10 vs. a Ag/AgCl reference electrode and startedat −0.4 V. An up and down-sweep is shown for each case.

FIG. 2: Selectively coated electrode array. The sub-50 nm gapsseparating opposing electrodes occur at the junction between the wideand narrow electrode features as indicated by the arrows. Electrodeslabelled 2, 4 and 6 were coated with oligonucleotide X using theselective desorption technique described and subsequently coloured usingthe anti-biotin antibody detection scheme described. The colour contrastbetween the electrodes (electrodes 2, 4 and 6 are significantly darkerthan electrodes 1, 3 and 5) shows that a very high degree of selectivecoating has been achieved across the nanometre sized gaps. The narrowcentral stripes are initially darker than the wide electrodes owing tothe metal layer being thinner.

FIG. 3: Scanning electron micrograph (SEM) of the junction regionbetween electrodes 1 and 4, of FIG. 2. The picture was taken after theanti-biotin antibody detection process. The inset is an enlarged view ofthe central region of the main picture and shows the gap to besignificantly less than 50 nm.

FIG. 4: Electrode arrays in which electrodes 1, 3 and 5 are coated witholigonucleotides Y and electrodes 2, 4 and 6 with oligonucleotides X.Arrows indicate the nanoscale gaps separating opposing electrodes. (a)Challenging the array with biotinylated oligonucelotides Y, followed bythe anti-biotin antibody detection process, electrodes 1, 3 and 5darken, confirming the presence of surface-bound oligonucelotides Y. (b)Challenging the array with biotinylated oligonucleotides X, followed bythe anti-biotin-antibody detection process, electrodes 2, 4 and 6darken, confirming the presence of surface-bound oligonucelotides X.

1. A method of forming coatings of at least two different coatingmolecules on at least two electrodes, the method comprising: (a)providing an array of at least two individually-addressable electrodes,(b) allowing a layer of a masking molecule to adsorb onto allelectrodes, (c) inducing electrochemical desorption of the maskingmolecule from at least one but not all electrodes to expose a first setof exposed electrodes, (d) allowing a first coating molecule to adsorbonto the first set of exposed electrodes, thereby generating a first setof coated electrodes, (e) exposing all electrodes, including the firstset of coated electrodes, to a masking molecule to allow adsorption ofthe masking molecule onto all electrodes, including the first set ofcoated electrodes, (f) inducing electrochemical desorption of maskingmolecule from a second set of electrodes to expose a second set ofexposed electrodes, (g) allowing a second coating molecule to adsorbonto the second set of exposed electrodes; wherein the first coatingmolecule and the second coating molecule each have a molecular weightgreater than or equal to 800 Da; and wherein the masking molecule has amolecular weight less than or equal to 500 Da.
 2. The method accordingto claim 1 in which the array comprises at least 10individually-addressable electrodes.
 3. The method according to claim 1comprising repeating steps (c) to (e) at least 8 times so as to formcoatings of at least 10 different coating molecules on at least 10different sets of electrodes.
 4. The method according to claim 1 inwhich the diameter of each electrode is not more than 50 μm.
 5. Themethod according to claim 1 in which the separation between electrodesis not more than 30 μm.
 6. The method according to claim 1 in which theelectrodes are metal electrodes and the masking molecules and thecoating molecules are thiolated.
 7. The method according to claim 1 inwhich the coating molecules are macromolecules having molecular weightof at least
 500. 8. The method according to claim 1 in which the coatingmolecules are oligonucleotides modified with a functional group capableof adsorbing onto the electrodes.
 9. The method according to claim 8additionally comprising providing nanoparticles functionalised witholigonucleotides complementary to the oligonucleotide coating moleculesand allowing the strands to hybridise.
 10. The method according to claim1 in which the coating molecules are polypeptides modified with afunctional group capable of adsorbing onto the electrodes.
 11. Themethod according to claim 1 in which step (b), step (d) or both alsocomprise application of an AC or DC electric field in order to induceorientation of the molecules being adsorbed.
 12. The method according toclaim 1 comprising controlling the potential of electrodes from whichdesorption is not required in steps (c), step (f) or both so as toprevent desorption from those electrodes.
 13. The method according toclaim 1 comprising application of an AC or DC potential to theelectrodes onto which adsorption is required in step (b), step (e), step(g) or any combination of these.
 14. The method of claim 1, wherein themasking molecule has a molecular weight less than or equal to 200 Da.15. The method of claim 1, wherein the masking molecule has a molecularweight less than or equal to 150 Da.
 16. The method of claim 1, whereinthe first coating molecule and the second coating molecule each have amolecular weight greater than or equal to 1000 Da.
 17. The method ofclaim 1, wherein the first coating molecule and the second coatingmolecule each have a molecular weight greater than or equal to 1500 Da.18. The method of claim 1, wherein the first coating molecule and thesecond coating molecule each have a molecular weight greater than orequal to 3000 Da.
 19. The method of claim 1, wherein the first coatingmolecule and the second coating molecule are each oligonucleotideshaving from 5 to 150 bases.
 20. The method of claim 1, wherein the firstcoating molecule and the second coating molecule are each proteins. 21.The method of claim 1, wherein the first coating molecule and the secondcoating molecule are each enzymes.
 22. The method of claim 1, whereinthe masking molecule is 6-mercapto-1-hexanol.
 23. The method of claim 1,wherein the first coating molecule is an oligonucleotide of sequenceCAGGATGGCGAACAACAAGA-thiol and the masking molecule is6-mercapto-1-hexanol.
 24. The method of claim 1, wherein the firstcoating molecule is an oligonucleotide of sequence AGGTCGCCGCCC-thioland the masking molecule is 6-mercapto-1-hexanol.
 25. The method ofclaim 1, wherein the first coating molecule is an oligonucleotide ofsequence CAGGATGGCGAACAACAAGA-thiol, the second coating molecule is anoligonucleotide of sequence AGGTCGCCGCCC-thiol, and the masking moleculeis 6-mercapto-1-hexanol.